ADAM10-Mediated Human Prion Protein Cleavage and Its Relationship with Neurodegenerative Diseases

Background

The endopeptidic processing of multifunctional proteins is crucial for regulating their physiological functions and plays a significant role in various pathological conditions. Prion protein (PrP), a widely expressed glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in the nervous system and is involved in various physiological tasks, suggesting its multifunctionality. However, its key pathological role in fatal and infectious neurodegenerative prion diseases, such as Creutzfeldt-Jakob Disease (CJD), has also been established. PrP leads to neuronal death and brain vacuolation through the templating and progressive misfolding and deposition of its pathogenic isoform, PrP^sc (prion protein scrapie). ADAM10, a disintegrin and metalloproteinase, mediates PrP cleavage and is considered a mechanism that regulates PrP function and participates in neurodegenerative diseases. Given the findings from studies on PrP cleavage in animal and in vitro models, this study aims to elucidate the biological significance of ADAM10 in the cleavage process and its relationship with neurodegenerative diseases by generating antibodies specifically detecting human cleaved PrP (shed PrP, sPrP).

Research Origin

This paper was published in 2024 by Feizhi Song, Valerija Kovac, Behnam Mohammadi, and Hermann C. Altmeppen in the journal “Acta Neuropathologica.” The authors are from several renowned research institutions, including University Medical Center Hamburg-Eppendorf, University of Ljubljana, and University Medical Center of Barcelona.

Research Process and Methods

Research Subjects and Sample Processing

The study collected various human and animal samples, including post-processed human tissue samples (such as paraffin-embedded tissue blocks and frozen samples), cerebrospinal fluid samples, and cell line samples. These samples were ethically approved and obtained and processed in laboratories of the respective institutions. Additionally, different types of transgenic mice (such as Prnp0/0 mice) were used for comparative studies to further validate the universality of the results.

Antibody Generation and Specificity Validation

Researchers first generated a polyclonal antibody, sprpy226, against PrP to detect sPrP presumed to be cleaved at Y226. They verified the specificity of these antibodies through experiments and initially tested them in human lung cancer cell line A549 and neuroblastoma cell line SH-SY5Y, confirming that the detected signals were dependent on ADAM10 activity rather than ADAM17.

Experimental Procedures

1. Structural Modeling and Cleavage Site Prediction: Using Pep-fold3 and FlexPepDock tools, the C-terminal sequence of human PrP was modeled, and potential ADAM10 cleavage sites were predicted.
2. Antibody Generation and Validation: Polyclonal antibody sprpy226 was generated in rabbits and validated through various biochemical experiments, including immunoblotting and ELISA.
3. Cell Line Testing: SH-SY5Y cells transfected with human PrP were used to test the generation of sPrP by employing ADAM10 inhibitors and antibodies 3F4 or 6D11.
4. Animal Model Validation: Experiments were conducted using Prnp0/0 mice and transgenic mice extensively expressing human PrP to verify the results in different biological backgrounds.

Main Research Results

1. Cleavage Site and ADAM10 Dependency:

   • Through modeling and experimental validation, the cleavage site of human PrP was determined at Y226, and the generated sPrP depended on ADAM10, not ADAM17.
   • Experiments showed that the ADAM10-specific inhibitor (such as GI254023X) could completely block sPrP signals, whereas general inhibitors (such as GW280264X) effective against both ADAM10 and ADAM17 did not.

2. Antibody Specificity Validation:

   • Using the generated polyclonal antibody sprpy226 and monoclonal antibody v5b2, the truncated form of human PrP (to Y226) and the intact tail form were detected. Immunoblotting and ELISA experiments verified that both antibodies had high specificity and affinity.
   • Further co-immunoprecipitation experiments showed that sprpy226 performed well in detecting denatured samples, whereas v5b2 performed better in native samples.

3. Feasibility of Antibody-Induced PrP Cleavage:

   • In the human nervous system, specific antibody binding can induce ADAM10-mediated PrP cleavage, suggesting that these antibodies can regulate PrP function in vitro.

Research Conclusions

This study, by generating specific antibodies, confirmed for the first time the physiological cleavage of human PrP at Y226 and demonstrated that this cleavage is entirely dependent on ADAM10. Further experiments indicated that this cleavage process has significant biological implications in neurodegenerative diseases, particularly in the pathological cases of PrP misfolding and accumulation in human and animal models. This research opens new potential pathways for the treatment of neurodegenerative diseases through PrP-targeted ligand-induced cleavage mechanisms.

Research Highlights

• Newly Discovered Cleavage Site: By combining structural prediction and experimental validation, the specific cleavage site of human PrP at Y226 was revealed for the first time.
• ADAM10 Dependency: The decisive role of ADAM10 in the PrP cleavage process, rather than other metalloproteinases, was clarified, providing a solid foundation for ADAM10-targeted therapeutic strategies.
• Specific Antibody Tools: The generated and validated high-specificity and high-affinity antibodies, sprpy226 and v5b2, can be widely used to detect and study sPrP in humans and related animals.

Significance and Application Value

This study is of significant scientific importance as it provides new insights into the role of prion proteins in neurodegenerative diseases. At the same time, by detecting and regulating the cleavage process of human PrP, developing new therapeutic strategies may become a reality, such as using specific antibodies to modulate PrP function to combat related diseases.

The study also offers the potential to develop alternative diagnostic biomarkers, as sPrP might become a readily accessible fluid biomarker, aiding in the early diagnosis and monitoring of neurodegenerative disease progression.